Methods summary: The program BoBro (Li et al., 2010) was used to identified cis-regulatory motifs in the 5’ DNA regulatory regions (DNA sequences up to 300 nt upstream from the transcriptional start site in the intergenic region and into the first 25 nt in the coding region) of R. pomeroyi DSS-3 genes that were transcriptionally perturbed under steady-state macronutrient limitations (Chan et al., 2012). Motifs with P-values <10-6 were considered significant (Li et al., 2010). These analyses resulted in the identification of 14 R. pomeroyi DSS-3 motifs. Using the program BoBro Based Searching/Scanning (BBS) tool, the 14 motifs were used as queries in subsequent genome-wide searches for orthologous motifs in the 5’ DNA regulatory regions associated with all protein-coding genes of 36 Roseobacter genomes, including R. pomeroyi, and in six non-Roseobacter (i.e., control) genomes: two distantly related marine bacteria in the Alphaproteobacteria lineage, two marine bacteria in Gammaproteobacteria, one marine bacterium in the Bacteroidetes lineage, and E. coli, a non-marine Gammaproteobacterium. Motif searches were considered significant if the resultant P-value was <10-7 and Z-score (Xie et al., 2005; Xie et al., 2007)>20. Bacterial genomes were downloaded from GenBank (as of May 2011).

Information for downloadable files: For each queried bacterial genome, two files are available: 1) a FASTA file contains the input 5’ DNA regulatory sequences used for genome-wide motif searches, and 2) a text file contains the resultant significant motifs. Header for the 5’ DNA regulatory sequence file has locus tag and the location of the queried regulatory sequence numbered according to the actual position in the genome. Header for the resultant significant motif file has this information: Gene, locus tag; Offset, location (in nt) of the motif start site from the transcriptional start site (+, upstream; -, downstream); Pos, start location of the motif sequence numbered according to the actual position in the genome; Motif, motif sequence; Score, see (Li et al., 2010); Intergenic, queried regulatory region length; Info, locus tag and the location of the motif sequence numbered according to the actual position in the genome.

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